ALS is an adult onset, progressive neurological disorder characterized by selective degeneration and death of motor neurons in the cortex and the spinal cord. In recent years, several RNA binding proteins have been linked to motor neuron disease, including senataxin, angiogenin, TDP-43 and FUS. These findings led to a paradigm shift in the current models for neuronal degeneration mechanisms and suggest that a significant component of ALS may be due to dysregulation of RNA metabolism. At present, TDP-43 has emerged as a common denominator for the majority of ALS cases known to date, however the mechanisms by which it causes neuronal degeneration remain poorly understood. The long-term goals of this research are to decipher the RNA-based mechanisms utilized by TDP-43 in the nervous system and to identify what aspects of RNA metabolism, specific protein partners and RNA targets are dysregulated by TDP-43 mutations linked to ALS. This exploratory proposal aims to test the hypothesis of RNA dysregulation in ALS by focusing on the physical and functional connections between TDP-43 and a repertoire of candidate RNA binding proteins. We will perform the proposed studies in a Drosophila model of ALS based on TDP-43 that we developed in our laboratory and which bears remarkable similarities to the human pathology. With help from collaborators our studies will be extended to human cells and tissue samples obtained from ALS patients. Preliminary data obtained from Drosophila and human cells show that TDP-43 forms a complex with FMRP, an RNA binding proteins with an established role in local translation and implicated in Fragile X syndrome. We also found that TDP-43 colocalizes with PABP in stress granules in motor neurons. In contrast to wild-type TDP-43, the A315T mutant, which has been linked to ALS in human patients, exhibits differential colocalization and genetic interactions with candidate RNA binding proteins including FMRP and PABP. We hypothesize that ALS stems in part from RNA dysregulation and propose to test this through a combination of molecular, electrophysiology, genetic and live imaging approaches. In Aim 1 we will establish the relationship between TDP-43 variants (wild-type and mutant) and several neuronal RNA granule components using colocalization, live trafficking studies, biochemical purifications and cellular fractionations under normal conditions or induced cellular stress. In Aim 2 we will use genetic interaction approaches in conjunction with a battery of phenotypic assays to establish the physiological significance of TDP-43's association with neuronal RNA granule components, including FMRP and established stress granule and P body markers. The proposed experiments will provide insights into what aspects of RNA regulation (e.g., stress granules assembly, composition, translation) are perturbed by disease causing mutations and will identify specific RNA binding proteins that modulate TDP-43's neurotoxicity in vivo. Given our extensive expertise in RNA based neuronal mechanisms and Drosophila genetics as well as the support from a team of expert collaborators we are uniquely positioned to test this novel and exciting hypothesis linking RNA dysregulation to neurodegeneration and to identify novel therapeutic targets for ALS.